Methods of cell culture

ABSTRACT

Polypeptide preparations having target levels of glycans, and methods of producing such polypeptide preparations using DMSO, are described.

FIELD OF THE INVENTION

The invention relates generally to cell culture methods.

BACKGROUND

Therapeutic polypeptides are an important class of therapeuticbiotechnology products, and therapeutic antibodies (including murine,chimeric, humanized and human antibodies and fragments thereof) accountfor the majority of therapeutic biologic products.

SUMMARY

In one aspect, the invention features a method of producing arecombinant protein preparation having a target value of one or more ofhigh mannose glycans and fucosylated glycans, the method comprising: (a)providing a cell genetically engineered to express a recombinantprotein; (b) culturing the cell in a culture medium comprising (e.g.,supplemented with) DMSO under conditions in which the cell expresses therecombinant protein; and (c) harvesting (e.g., purifying or isolatingfrom the cell and/or culture medium) a preparation of the recombinantprotein produced by the cell, wherein the preparation has the targetvalue of the one or more of high mannose glycans and fucosylatedglycans. In some embodiments, the culture medium comprises DMSO for atime and in an amount effective to modify (e.g., increase or decrease)one or more of the high mannose glycans and fucosylated glycans of therecombinant protein.

In some embodiments, the culture medium comprises at least about 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%,2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%,3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%. 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or5% DMSO. In some embodiments, the culture medium comprises about 0.1% to5% DMSO, about 0.1% to about 1% DMSO, about 1% to about 2% DMSO, about2% to about 3% DMSO, about 3% to about 4% DMSO, about 4% to about 5%DMSO, about 0.1% to about 2.5% DMSO, about 2.5% to about 5% DMSO, about0.1% to about 2% DMSO, about 0.1% to about 3% DMSO, about 0.1% to about4% DMSO, about 1% to about 5% DMSO, about 2% to about 5% DMSO, or about3% to about 5% DMSO.

In some embodiments, the target value is a level of one or more of highmannose glycans and fucosylated glycans in a reference therapeuticproduct. In some embodiments, the target value is a level of one or moreof high mannose glycans and fucosylated glycans in a referencetherapeutic antibody product. In some embodiments, the target value is apredetermined pharmaceutical product specification or a quality controlcriterion for a pharmaceutical preparation, e.g., a Certificate ofAnalysis (CofA), a Certificate of Testing (CofT), or a Master BatchRecord. In some embodiments, the product specification is a productdescription in an FDA label, a Physician's Insert, a USP monograph, oran EP monograph.

In some embodiments, the reference therapeutic product is selected fromthe group consisting of: abatacept, abciximab, adalimumab, aflibercept,alefacept, alemtuzumab, basiliximab, bevacizumab, belatacept,certolizumab, cetuximab, daclizumab, eculizumab, efalizumab,entanercept, gemtuzumab, ibritumomab, infliximab, muromonab-CD3,natalizumab, omalizumab, palivizumab; panitumumab, ranibizumab,rilonacept, rituximab, tositumomab, and trastuzumab.

In some embodiments, the target value is one or more of: (a) at leastabout 0.1% to about 20% high mannose glycans, e.g., at least about 0.1%,about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, or more, high mannose glycans; and (b) at least about 70% to100% fucosylated glycans, e.g., at least about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or about 100% fucosylated glycans.High mannose glycans can be, e.g., HM3, HM4, HM5, HM6, HM7, HM8, HM9, orcombinations thereof.

In some embodiments, the target value of the one or more of high mannoseglycans and fucosylated glycans is higher than a corresponding level ina preparation produced by culturing the cell in the medium notcomprising DMSO. In some embodiments, the target value is higher thanthe corresponding level by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, ormore, of the corresponding level.

In some embodiments, the method further comprises evaluating the levelof one or more of high mannose glycans and fucosylated glycans in therecombinant protein preparation. In some embodiments, the method furthercomprises recording the level in a print or computer-readable medium,e.g., in a test report, Material Safety Data Sheet (MSDS), batch record,Certificate of Testing (CofT) or Certificate of Analysis (CofA).

In another aspect, the invention features a method of producing arecombinant protein preparation, the method comprising: (a) providing atarget value of one or more of high mannose glycans and fucosylatedglycans; (b) providing a cell genetically engineered to express arecombinant protein; (c) culturing the cell in a culture mediumcomprising (e.g., supplemented with) DMSO under conditions in which thecell expresses the recombinant protein; (d) harvesting a preparation ofthe recombinant protein produced by the cell; and (e) processing (e.g.,one or more of formulating, filling into a container, labeling,packaging) the preparation into a drug product if the preparation meetsthe target value of the one or more of high mannose glycans andfucosylated glycans. In some embodiments, the culture medium comprisesDMSO for a time and in an amount effective to modify (e.g., increase ordecrease) one or more of the high mannose glycans and fucosylatedglycans of the recombinant protein.

In some embodiments, the method further comprises evaluating the levelof one or more of high mannose glycans and fucosylated glycans in therecombinant protein preparation. In some embodiments, the method furthercomprises recording the level in a print or computer-readable medium,e.g., in a test report, Material Safety Data Sheet (MSDS), batch record,or Certificate of Testing (CofT) or Certificate of Analysis (CofA).

In some embodiments, the culture medium comprises at least about 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%,2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%,3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%. 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or5% DMSO. In some embodiments, the culture medium comprises about 0.1% to5% DMSO, about 0.1% to about 1% DMSO, about 1% to about 2% DMSO, about2% to about 3% DMSO, about 3% to about 4% DMSO, about 4% to about 5%DMSO, about 0.1% to about 2.5% DMSO, about 2.5% to about 5% DMSO, about0.1% to about 2% DMSO, about 0.1% to about 3% DMSO, about 0.1% to about4% DMSO, about 1% to about 5% DMSO, about 2% to about 5% DMSO, or about3% to about 5% DMSO.

In some embodiments, the target value is a level of one or more of highmannose glycans and fucosylated glycans in a reference therapeuticproduct. In some embodiments, the target value is a level of one or moreof high mannose glycans and fucosylated glycans in a referencetherapeutic antibody product. In some embodiments, the target value is apredetermined pharmaceutical product specification or a quality controlcriterion for a pharmaceutical preparation, e.g., a Certificate ofAnalysis (CofA), a Certificate of Testing (CofT), or a Master BatchRecord. In some embodiments, the product specification is a productdescription in an FDA label, a Physician's Insert, a USP monograph, oran EP monograph.

In some embodiments, the reference therapeutic product is selected fromthe group consisting of: abatacept, abciximab, adalimumab, aflibercept,alefacept, alemtuzumab, basiliximab, bevacizumab, belatacept,certolizumab, cetuximab, daclizumab, eculizumab, efalizumab,entanercept, gemtuzumab, ibritumomab, infliximab, muromonab-CD3,natalizumab, omalizumab, palivizumab; panitumumab, ranibizumab,rilonacept, rituximab, tositumomab, and trastuzumab.

In some embodiments, the target value is one or more of: (a) at leastabout 0.1% to about 20% high mannose glycans, e.g., at least about 0.1%,about 0.5%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, or more, high mannose glycans; and (b) at least about 70% to100% fucosylated glycans, e.g., at least about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or about 100% fucosylated glycans.High mannose glycans can be, e.g., HM3, HM4, HM5, HM6, HM7, HM8, HM9, orcombinations thereof.

In some embodiments, the target value of the one or more of high mannoseglycans and fucosylated glycans is higher than a corresponding level ina preparation produced by culturing the cell in the medium notcomprising DMSO. In some embodiments, the target value is higher thanthe corresponding level by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, ormore, of the corresponding level.

In another aspect, the invention features a method of increasing a levelof one or more of high mannose glycans, sialylated glycans, and3,3,1,0,0 glycans in a recombinant protein preparation, the methodcomprising: (a) providing a cell genetically engineered to express arecombinant protein; (b) culturing the cell in a culture mediumcomprising (e.g., supplemented with) DMSO under conditions in which thecell expresses the recombinant protein; and (c) harvesting a preparationof the recombinant protein produced by the cell, wherein the preparationhas an increased level of one or more of high mannose glycans,sialylated glycans, and 3,3,1,0,0 glycans relative to a correspondinglevel in a preparation of the recombinant protein produced by culturingthe cell in the medium not comprising DMSO. In some embodiments, theculture medium comprises DMSO for a time and in an amount effective toincrease one or more of the high mannose glycans, sialylated glycans,and 3,3,1,0,0 glycans of the recombinant protein. In some embodiments,the method further comprises processing, (e.g., one or more offormulating, filling into a container, labeling, packaging) thepreparation into a drug product if the preparation meets a target valueof one or more of high mannose glycans, sialylated glycans, and3,3,1,0,0 glycans.

In some embodiments, the culture medium comprises at least about 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%,2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%,3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%. 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or5% DMSO. In some embodiments, the culture medium comprises about 0.1% to5% DMSO, about 0.1% to about 1% DMSO, about 1% to about 2% DMSO, about2% to about 3% DMSO, about 3% to about 4% DMSO, about 4% to about 5%DMSO, about 0.1% to about 2.5% DMSO, about 2.5% to about 5% DMSO, about0.1% to about 2% DMSO, about 0.1% to about 3% DMSO, about 0.1% to about4% DMSO, about 1% to about 5% DMSO, about 2% to about 5% DMSO, or about3% to about 5% DMSO.

In some embodiments, the method further comprises measuring a level ofthe one or more of high mannose glycans, sialylated glycans, and3,3,1,0,0 glycans in the recombinant protein preparation. In someembodiments, the method further comprises recording the level in a printor computer-readable medium, e.g., in a test report, Material SafetyData Sheet (MSDS), batch record, Certificate of Testing (CofT) orCertificate of Analysis (CofA).

In some embodiments, the increased level of the one or more of highmannose glycans, sialylated glycans, and 3,3,1,0,0 glycans is higherthan the corresponding level by at least about 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%,500%, or more, of the corresponding level. In some embodiments, theincreased level is one or more of: (a) at least about 0.1% to about 20%high mannose glycans, e.g., at least about 0.1%, about 0.5%, about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about15%, about 16%, about 17%, about 18%, about 19%, about 20%, or more,high mannose glycans; (b) at least about 70% to 100% fucosylatedglycans, e.g., at least about 70%, about 75%, about 80%, about 85%,about 90%, about 95%, or about 100% fucosylated glycans; and (c) atleast about 0.1% to about 90% sialylated glycans, e.g., at least about20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% sialylated glycans. Highmannose glycans can be, e.g., HM3, HM4, HM5, HM6, HM7, HM8, HM9, orcombinations thereof.

In another aspect, the invention features a method of decreasing a levelof one or more of high mannose glycans, sialylated glycans, and3,3,1,0,0 glycans in a recombinant protein preparation, the methodcomprising: (a) providing a cell genetically engineered to express arecombinant protein; (b) culturing the cell in a culture mediumcomprising a reduced level of DMSO under conditions in which the cellexpresses the recombinant protein; and (c) harvesting a preparation ofthe recombinant protein produced by the cell, wherein the preparationhas a decreased level of one or more of high mannose glycans, sialylatedglycans, and 3,3,1,0,0 glycans relative to a corresponding level in apreparation of the recombinant protein produced by culturing the cell inthe medium not comprising the reduced level of DMSO. In someembodiments, the culture medium comprises the reduced level of DMSO fora time and in an amount effective to decrease a level of one or more ofthe high mannose glycans, sialylated glycans, and 3,3,1,0,0 glycans ofthe recombinant protein. In some embodiments, the method furthercomprises processing, (e.g., one or more of formulating, filling into acontainer, labeling, packaging) the preparation into a drug product ifthe preparation meets a target value of one or more of high mannoseglycans, sialylated glycans, and 3,3,1,0,0 glycans.

In some embodiments, the medium comprises less than about 5%, 4.9%,4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%,3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%,2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%,1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%;or 0% DMSO.

In some embodiments, the method further comprises measuring a level ofthe one or more of high mannose glycans, sialylated glycans, and3,3,1,0,0 glycans in the recombinant protein preparation. In someembodiments, the method further comprises recording the level in a printor computer-readable medium, e.g., in a test report, Material SafetyData Sheet (MSDS), batch record, Certificate of Testing (CofT) orCertificate of Analysis (CofA).

In some embodiments, the decreased level of the one or more of highmannose glycans, sialylated glycans, and 3,3,1,0,0 glycans is lower thanthe corresponding level by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or 100%, of the corresponding level. In someembodiments, the decreased level is one or more of: (a) less than about20% high mannose glycans; (b) less than about 90% fucosylated glycans;and (c) less than about 90% sialylated glycans. High mannose glycans canbe, e.g., HM3, HM4, HM5, HM6, HM7, HM8, HM9, or combinations thereof.

In another aspect, the invention features a method of decreasing a levelof one or more fucosylated glycans in a recombinant protein preparation,the method comprising: (a) providing a cell genetically engineered toexpress a recombinant protein; (b) culturing the cell in a culturemedium comprising (e.g., supplemented with) DMSO under conditions inwhich the cell expresses the recombinant protein; and (c) harvesting apreparation of the recombinant protein produced by the cell, wherein thepreparation has a decreased level of one or more fucosylated glycansrelative to a corresponding level in a preparation of the recombinantprotein produced by culturing the cell in the medium not comprisingDMSO. In some embodiments, the culture medium comprises DMSO for a timeand in an amount effective to decrease the level of one or morefucosylated glycans of the recombinant protein. In some embodiments, themethod further comprises processing, (e.g., one or more of formulating,filling into a container, labeling, packaging) the preparation into adrug product if the preparation meets a target value of one or morefucosylated glycans.

In some embodiments, the culture medium comprises at least about 0.1%,0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%,2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%,3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%. 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, or5% DMSO. In some embodiments, the culture medium comprises about 0.1% to5% DMSO, about 0.1% to about 1% DMSO, about 1% to about 2% DMSO, about2% to about 3% DMSO, about 3% to about 4% DMSO, about 4% to about 5%DMSO, about 0.1% to about 2.5% DMSO, about 2.5% to about 5% DMSO, about0.1% to about 2% DMSO, about 0.1% to about 3% DMSO, about 0.1% to about4% DMSO, about 1% to about 5% DMSO, about 2% to about 5% DMSO, or about3% to about 5% DMSO.

In some embodiments, the method further comprises measuring a level offucosylated glycans in the recombinant protein preparation. In someembodiments, the method further comprises recording the level in a printor computer-readable medium, e.g., in a test report, Material SafetyData Sheet (MSDS), batch record, Certificate of Testing (CofT) orCertificate of Analysis (CofA).

In some embodiments, the decreased level of the fucosylated glycans islower than the corresponding level by at least about 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95%, or 100%, of the corresponding level. Insome embodiments, the decreased level is less than about 100%fucosylated glycans. In some embodiments, the one or more fucosylatedglycans comprise G0F glycans.

In another aspect, the invention features a method of increasing a levelof one or more fucosylated glycans in a recombinant protein preparation,the method comprising: (a) providing a cell genetically engineered toexpress a recombinant protein; (b) culturing the cell in a culturemedium comprising a reduced level of DMSO under conditions in which thecell expresses the recombinant protein; and (c) harvesting a preparationof the recombinant protein produced by the cell, wherein the preparationhas an increased level of one or more fucosylated glycans relative to acorresponding level in a preparation of the recombinant protein producedby culturing the cell in the medium not comprising the reduced level ofDMSO. In some embodiments, the culture medium comprises a reduced levelof DMSO for a time and in an amount effective to increase the level ofone or more fucosylated glycans of the recombinant protein. In someembodiments, the method further comprises processing, (e.g., one or moreof formulating, filling into a container, labeling, packaging) thepreparation into a drug product if the preparation meets a target valueof one or more fucosylated glycans.

In some embodiments, the medium comprises less than about 5%, 4.9%,4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%,3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%,2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%,1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%;or 0% DMSO.

In some embodiments, the method further comprises measuring a level offucosylated glycans in the recombinant protein preparation. In someembodiments, the method further comprises recording the level in a printor computer-readable medium, e.g., in a test report, Material SafetyData Sheet (MSDS), batch record, Certificate of Testing (CofT) orCertificate of Analysis (CofA).

In some embodiments, the increased level of the fucosylated glycans ishigher than the corresponding level by at least about 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%,450%, 500%, or more, of the corresponding level. In some embodiments,the increased level is at least about 70% to 100% fucosylated glycans,e.g., at least about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or about 100% fucosylated glycans. In some embodiments, theone or more fucosylated glycans comprise G0F glycans.

In some aspects described herein, the culturing step comprises a firststage and a second stage. In some embodiments, the first stage comprisesculturing the cell in a first culture medium comprising a first level ofDMSO, and the second stage comprises culturing the cell in a secondculture medium comprising a second level of DMSO. In some embodiments,the first culture medium comprises a reduced level of (e.g., does notcomprise) DMSO relative to the second culture medium and the secondculture medium comprises an elevated level of DMSO (e.g., about 1% toabout 5% DMSO) relative to the first culture medium. In someembodiments, the first culture medium comprises an elevated level ofDMSO (e.g., about 1% to about 5% DMSO), and the second culture mediumcomprises a reduced level of (e.g., does not comprise) DMSO.

In some embodiments, the first stage comprises culturing the cell in thefirst culture medium for about 1 to about 8 days, e.g., 1-7, 1-6, 1-5days. In some embodiments, the second stage comprises culturing the cellin the second culture medium for about 1 to about 12 days, e.g., 1-10,1-9, 1-8, 1-7, 1-6 days. In some embodiments, the first stage is agrowth stage. In some embodiments, the second stage is a productionstage.

In some aspects described herein, the culture medium further comprisesone or more of lysine, cysteine, ammonium, manganese, copper, cobalt,putrescine, a peptone, glucose, galactose, glucosamine, glutamine, alipid (e.g., cholesterol), and dextran sulfate.

In some aspects described herein, the cell is a mammalian cell. In someembodiments, the mammalian cell is a CHO (e.g., CHO-K1, DG44, CHO-DXB11,CHOK1SV, CHO-S) Vero, BHK, HeLa, COS, MDCK, or HEK-293 cell.

In some aspects described herein, the recombinant protein is arecombinant therapeutic product. In some embodiments, the recombinantprotein is a recombinant therapeutic antibody product. In someembodiments, the recombinant protein is a recombinant therapeutic fusionprotein. In some embodiments, the recombinant protein is abatacept,abciximab, adalimumab, aflibercept, alefacept, alemtuzumab, basiliximab,bevacizumab, belatacept, certolizumab, cetuximab, daclizumab,eculizumab, efalizumab, entanercept, gemtuzumab, ibritumomab,infliximab, muromonab-CD3, natalizumab, omalizumab, palivizumab;panitumumab, ranibizumab, rilonacept, rituximab, tositumomab, andtrastuzumab.

In some aspects described herein, the conditions in which cells (e.g.,mammalian cells) express recombinant proteins comprise (i) a mediumhaving a pH of about 6, about 6.5, about 6.6, about 6.7, about 6.8,about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about7.5, or about 8; (ii) a temperature of about 25° C., about 26° C., about27° C., about 28° C., about 29° C., about 30° C., about 31° C., about32° C., about 33° C., about 34° C., about 35° C., about 36° C., about37° C., about 38° C., about 39° C., or about 40° C.; and/or (iii) aculture volume of about 100 mL, about 200 mL, about 300 mL, about 400mL, about 500 mL, about 600 mL, about 700 mL, about 800 mL, about 900mL, about 1 L, about 2 L, about 3 L, about 5 L, about 10 L, about 20 L,about 30 L, about 40 L, about 50 L, about 100 L, about 200 L, about 300L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L,about 900 L, about 1000 L, 5,000 L, 10,000 L, 20,000 L, or more.

In another aspect, the invention features a preparation of a recombinantprotein produced using a method described herein.

In another aspect, the invention features a method of producing arecombinant therapeutic antibody preparation (e.g., abciximab,adalimumab, alemtuzumab, basiliximab, bevacizumab, certolizumab,cetuximab, daclizumab, eculizumab, efalizumab, gemtuzumab, ibritumomab,infliximab, muromonab-CD3, natalizumab, omalizumab, palivizumab,panitumumab, ranibizumab, rituximab, tositumomab, or trastuzumab), themethod comprising: (a) providing a target value (e.g., a predeterminedpharmaceutical product specification or a quality control criterion fora pharmaceutical preparation, e.g., a Certificate of Analysis (CofA), aCertificate of Testing (CofT), or a Master Batch Record of a referencetherapeutic antibody product) of one or more of high mannose glycans(e.g., HM3, HM4, HM5, HM6, HM7, HM8, HM9, or combinations thereof) andfucosylated glycans; (b) providing a CHO cell genetically engineered toexpress a recombinant antibody; (c) culturing the cell in a culturemedium comprising 0.1% to 5% DMSO (e.g., at least about 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%,1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%,2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%,3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%. 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5% DMSO,or about 0.1% to 5% DMSO, about 0.1% to about 1% DMSO, about 1% to about2% DMSO, about 2% to about 3% DMSO, about 3% to about 4% DMSO, about 4%to about 5% DMSO, about 0.1% to about 2.5% DMSO, about 2.5% to about 5%DMSO, about 0.1% to about 2% DMSO, about 0.1% to about 3% DMSO, about0.1% to about 4% DMSO, about 1% to about 5% DMSO, about 2% to about 5%DMSO, or about 3% to about 5% DMSO) under conditions in which the cellexpresses the recombinant antibody; (d) harvesting (e.g., purifying orisolating from the cell and/or culture medium) a preparation of therecombinant antibody produced by the cell; and (e) formulating (e.g.,one or more of formulating, filling into a container, labeling,packaging) the preparation into a drug product if the preparation meetsthe target value of the one or more of high mannose glycans andfucosylated glycans.

BRIEF DESCRIPTION OF THE DRAWINGS

The present teachings described herein will be more fully understoodfrom the following description of various illustrative embodiments, whenread together with the accompanying drawings. It should be understoodthat the drawings described below are for illustration purposes only andare not intended to limit the scope of the present teachings in any way.

FIG. 1 is a schematic illustration of an IgG antibody molecule.

FIG. 2A is a graphic representation of G0F glycan levels in preparationsof a model antibody from cells grown in medium with or without DMSO.FIG. 2B is a graphic representation of the two G1F isomers (indicated asG1Fa and G1Fb), or G2F glycan levels in preparations of a model antibodyfrom cells grown in medium with or without DMSO.

FIG. 3A is a graphic representation of total high mannose glycan levelsin preparations of a model antibody from cells grown in medium with orwithout DMSO. FIG. 3B is a graphic representation of high mannose 6(HM6) glycan level, high mannose 5 (HM5) glycan level, or high mannose 3and 4 (HM3 and HM4) glycan level in preparations of a model antibodyfrom cells grown in medium with or without DMSO.

FIG. 4A is a graphic representation of fucosylated glycan levels inpreparations of a model antibody from cells grown in medium with orwithout DMSO. FIG. 4B is a graphic representation of afucosylated glycanlevels in preparations of a model antibody from cells grown in mediumwith or without DMSO.

FIG. 5A is a graphic representation of total sialylated glycan levels inpreparations of a model antibody from cells grown in medium with orwithout DMSO. FIG. 5B is a graphic representation of 3,5,1,1,0sialylated glycan levels in preparations of a model antibody from cellsgrown in medium with or without DMSO.

FIG. 6 depicts structural illustrations of 3,3,1,0,0 and 3,5,1,1,0glycans (triangle=fucose; dark gray circles=mannose; squares=N-acetylglucosamine; light gray circle (between square and diamond)=galactose;diamond=sialic acid). “2AB” is a dye conjugated to the end of the glycanthat is linked to a polypeptide.

DETAILED DESCRIPTION

The inventors have discovered that preparations of polypeptides (e.g.,antibodies) having targeted levels of glycans (e.g., high mannoseglycans, fucosylated glycans, and/or sialylated glycans) can be producedfrom cells cultured in a medium having dimethylsulfoxide (DMSO), e.g., aparticular level of DMSO effective to cause such effect. Surprisingly,culturing the cells in medium comprising DMSO does not affect cellgrowth, cell viability, or titer. The present disclosure encompassespreparations of polypeptides (e.g., antibodies) having targeted levelsof glycans (e.g., high mannose glycans, fucosylated glycans, and/orsialylated glycans), methods of making such polypeptides (e.g.,antibodies), and methods of using such polypeptides (e.g., antibodies).

DEFINITIONS

As used herein, “purified” (or “isolated”) refers to a nucleic acidsequence (e.g., a polynucleotide) or an amino acid sequence (e.g., apolypeptide) that is substantially free of other components. In someembodiments, a purified polynucleotide or purified polypeptide isremoved or separated from other components present in its naturalenvironment. For example, an isolated polypeptide is one that isseparated from other components of a cell in which it was produced(e.g., the endoplasmic reticulum or cytoplasmic proteins and RNA). Anisolated polynucleotide is one that is separated from other nuclearcomponents (e.g., histones) and/or from upstream or downstream nucleicacid sequences. An isolated nucleic acid sequence or amino acid sequencecan be at least 60% free, or at least 75% free, or at least 90% free, orat least 95% free from other components present in natural environmentof the indicated nucleic acid sequence or amino acid sequence.

As used herein, “polynucleotide” (or “nucleotide sequence” or “nucleicacid molecule”) refers to an oligonucleotide, nucleotide, orpolynucleotide, and fragments or portions thereof, and to DNA and RNA ofgenomic or synthetic origin, which may be single- or double-stranded,and represent the sense or anti-sense strand.

As used herein, “polypeptide” (or “amino acid sequence” or “protein”)refers to an oligopeptide, peptide, polypeptide, or protein sequence,and fragments or portions thereof, and to naturally occurring orsynthetic molecules. “Amino acid sequence” and like terms, such as“polypeptide” or “protein”, are not meant to limit the indicated aminoacid sequence to the complete, native amino acid sequence associatedwith the recited protein molecule.

The term “pharmaceutically effective amount” or “therapeuticallyeffective amount” refers to an amount (e.g., dose) effective in treatinga patient, having a disorder or condition described herein. It is alsoto be understood herein that a “pharmaceutically effective amount” maybe interpreted as an amount giving a desired therapeutic effect, eithertaken in one dose or in any dosage or route, taken alone or incombination with other therapeutic agents.

The term “treatment” or “treating”, as used herein, refers toadministering a therapy in an amount, manner, and/or mode effective toimprove a condition, symptom, or parameter associated with a disorder orcondition or to prevent or reduce progression of a disorder orcondition, to a degree detectable to one skilled in the art. Aneffective amount, manner, or mode can vary depending on the subject andmay be tailored to the subject.

As used herein, the term “antibody” refers to a polypeptide thatincludes at least one immunoglobulin variable region, e.g., an aminoacid sequence that provides an immunoglobulin variable domain orimmunoglobulin variable domain sequence. For example, an antibody caninclude a heavy (H) chain variable region (abbreviated herein as VH),and a light (L) chain variable region (abbreviated herein as VL). Inanother example, an antibody includes two heavy (H) chain variableregions and two light (L) chain variable regions. The term “antibody”encompasses antigen-binding fragments of antibodies (e.g., single chainantibodies, Fab, F(ab′)₂, Fd, Fv, and dAb fragments) as well as completeantibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD,IgM (as well as subtypes thereof). The light chains of theimmunoglobulin can be of types kappa or lambda. In some embodiments, anantibody includes an Fc region. In some embodiments, an antibody is atherapeutic antibody.

As used herein, the term “Fc region” refers to a dimer of two “Fcpolypeptides”, each “Fc polypeptide” comprising the constant region ofan antibody excluding the first constant region immunoglobulin domain.In some embodiments, an “Fc region” includes two Fc polypeptides linkedby one or more disulfide bonds, chemical linkers, or peptide linkers.“Fc polypeptide” refers to the last two constant region immunoglobulindomains of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM, and may also include part or allof the flexible hinge N-terminal to these domains. For IgG, “Fcpolypeptide” comprises immunoglobulin domains Cgamma2 (Cγ2) and Cgamma3(Cγ3) and the lower part of the hinge between Cgamma1 (Cγ1) and Cγ2.Although the boundaries of the Fc polypeptide may vary, the human IgGheavy chain Fc polypeptide is usually defined to comprise residuesstarting at T223 or C226 or P230, to its carboxyl-terminus, wherein thenumbering is according to the EU index as in Kabat et al. (1991, NIHPublication 91-3242, National Technical Information Services,Springfield, Va.). For IgA, Fc polypeptide comprises immunoglobulindomains Calpha2 (Cα2) and Calpha3 (Cα3) and the lower part of the hingebetween Calpha1 (Cα1) and Cα2. An Fc region can be synthetic,recombinant, or generated from natural sources such as IVIG.

As used herein, a “glycan” is a sugar. Glycans can be monomers orpolymers of sugar residues, but typically contain at least three sugars,and can be linear or branched. A glycan may include natural sugarresidues (e.g., glucose, N-acetylglucosamine, N-acetyl neuraminic acid,galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.)and/or modified sugars (e.g., 2′-fluororibose, 2′-deoxyribose,phosphomannose, 6′-sulfo N-acetylglucosamine, etc). The term “glycan”includes homo and heteropolymers of sugar residues. The term “glycan”also encompasses a glycan component of a glycoconjugate (e.g., of aglycoprotein, glycolipid, proteoglycan, etc.). The term also encompassesfree glycans, including glycans that have been cleaved or otherwisereleased from a glycoconjugate.

As used herein, a “high mannose glycan” refers to a glycan that includesat least 3 mannose sugar residues and that terminates in a mannose on anon-reducing end of the glycan. In some embodiments, a “high mannoseglycan” includes at least 4, 5, 6, 7, 8, 9, 10, 11, or 12 mannose sugarresidues.

As used herein, a “sialylated glycan” refers to a glycan that includesat least 1 sialic acid. In some embodiments, a sialylated glycanincludes at least 1, 2, 3, or 4 sialic acids. In some embodiments, asialylated glycan is a monosialylated glycan (e.g., a branched glycanmonosialylated on an α1-3 arm of the branched glycan (e.g., with aNeuAc-α2,6-Gal terminal linkage)), and/or a disialylated glycan (e.g., abranched glycan sialylated on both an α1-3 arm and an α1-6 arm of thebranched glycan).

As used herein, the term “glycoprotein preparation” refers to a set ofindividual glycoprotein molecules, each of which comprises a polypeptidehaving a particular amino acid sequence (which amino acid sequenceincludes at least one glycosylation site) and at least one glycancovalently attached to the at least one glycosylation site. Individualmolecules of a particular glycoprotein within a glycoprotein preparationtypically have identical amino acid sequences but may differ in theoccupancy of the at least one glycosylation sites and/or in the identityof the glycans linked to the at least one of the glycosylation sites.That is, a glycoprotein preparation may contain only a single glycoformof a particular glycoprotein, but more typically contains a plurality ofglycoforms. Different preparations of the same glycoprotein may differin the identity of glycoforms present (e.g., a glycoform that is presentin one preparation may be absent from another) and/or in the relativeamounts of different glycoforms.

The term “glycoform” is used herein to refer to a particular form of aglycoprotein. That is, when a glycoprotein includes a particularpolypeptide that has the potential to be linked to different glycans orsets of glycans, then each different version of the glycoprotein (i.e.,where the polypeptide is linked to a particular glycan or set ofglycans) is referred to as a “glycoform”.

“Reference glycoprotein”, as used herein, refers to a glycoproteinhaving substantially the same amino acid sequence as (e.g., having about95-100% identical amino acids of) a glycoprotein described herein, e.g.,a glycoprotein to which it is compared. In some embodiments, a referenceglycoprotein is a therapeutic glycoprotein described herein, e.g., anFDA approved therapeutic glycoprotein.

As used herein, an “N-glycosylation site of an Fc region” refers to anamino acid residue within an Fc region to which a glycan is N-linked.

“Target value”, as used herein, refers to a predetermined level of oneor more particular glycans, such as high mannose glycans, fucosylatedglycans, and/or sialylated glycans. In some embodiments, a target valueis an absolute value. In some embodiments, a target value is a relativevalue. In some embodiments, a target value is a level of one or moreparticular glycans, such as high mannose glycans (e.g., HM3, HM4, HM5,HM6, HM7, HM8, HM9, or combinations), fucosylated glycans (e.g., G0F,G1F, G2F, or combinations), and/or sialylated glycans (e.g.,monosialylated, disialylated, or combinations), in a referenceglycoprotein product or described in a specification or master batchrecord for a pharmaceutical product.

In some embodiments, a target value refers to an absolute level of(e.g., number of moles of) one or more glycans (e.g., high mannoseglycans (e.g., one or more species of high mannose glycans), fucosylatedglycans (e.g., one or more species of fucosylated glycans), and/orsialylated glycans (e.g., one or more species of sialylated glycans) ina glycoprotein preparation. In some embodiments, a target value refersto a level of one or more glycans (e.g., high mannose glycans (e.g., oneor more species of high mannose glycans), fucosylated glycans (e.g., oneor more species of fucosylated glycans), and/or sialylated glycans(e.g., one or more species of sialylated glycans) in a glycoproteinpreparation relative to total level of glycans in the glycoproteinpreparation. In some embodiments, a target value is expressed as a“percent”, which refers to the number of moles of one or more glycans(e.g., Fc glycans) relative to total moles of glycans (e.g., Fc glycans)in a glycoprotein preparation. In some embodiments, “percent” refers tothe number of moles of one or more PNGase F-released Fc glycans relativeto total moles of PNGase F-released Fc glycans detected.

Cells

Any host cell that can be used to express a polypeptide of interest(e.g., an antibody) can be used in the methods described herein. Thecells can be genetically engineered to contain a recombinant nucleicacid sequence, e.g., a gene, that encodes a polypeptide of interest(e.g., an antibody). For example, useful cells can express a recombinantpolypeptide. Recombinant expression of a gene encoding a polypeptide caninclude construction of an expression vector containing a polynucleotidethat encodes the polypeptide. Once a polynucleotide has been obtained, avector for the production of the polypeptide can be produced byrecombinant DNA technology using techniques known in the art. Knownmethods can be used to construct expression vectors containingpolypeptide coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination.

An expression vector can be transferred to a host cell by conventionaltechniques, and the transfected cells can then be cultured byconventional techniques, modified in accordance with the presentdisclosure, to produce a recombinant polypeptide. A variety of hostexpression vector systems can be used (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems (e.g., genetically engineeredhost expression systems) can be used to produce polypeptides (e.g.,antibodies) and, where desired, subsequently purified. Such hostexpression systems include, but are not limited to, yeast (e.g.,Saccharomyces and Pichia) transformed with recombinant yeast expressionvectors containing polypeptide coding sequences; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing polypeptide coding sequences; plant cell systems infectedwith recombinant virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinantplasmid expression vectors (e.g., Ti plasmid) containing polypeptidecoding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293,NS0, and 3T3 cells) harboring recombinant expression constructscontaining promoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter).

For expression in mammalian host cells, viral-based expression systemscan be utilized (see, e.g., Logan et al., 1984, Proc. Natl. Acad. Sci.USA 8:355-359). The efficiency of expression can be enhanced by theinclusion of appropriate transcription enhancer elements, transcriptionterminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol.153:516-544).

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the polypeptide (e.g., antibody)expressed. Such cells include, for example, established mammalian celllines and insect cell lines, animal cells, fungal cells, and yeastcells. Mammalian host cells include, but are not limited to, CHO, Vero,BHK, HeLa, COS, MDCK, HEK-293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20,T47D, NS0 (a murine myeloma cell line that does not endogenously produceany immunoglobulin chains), CRL7O3O, HsS78Bst cells, PER.C6, SP2/0-Ag14,and hybridoma cells. Additional, nonlimiting examples of animal ormammalian host cells include Chinese hamster ovary cells (CHO), such asCHO-K1 (ATCC CCL-61), DG44 (Chasin et al., 1986, Som. Cell Molec.Genet., 12:555-556; and Kolkekar et al., 1997, Biochem.,36:10901-10909), CHO-DXB11 (G. Urlaub and L. A. Chasin, 1980 Proc. Natl.Acad. Sci., 77: 4216-4220. L. H. Graf, and L. A. Chasin 1982, Molec.Cell. Biol., 2: 93-96), CHO-K1 Tet-On cell line (Clontech), CHOdesignated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13(GEIMG, Genova, IT), CHO clone B (GEIMG, Genova, IT), CHO-K1/SFdesignated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK), RR-CHOK1designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK), CHOK1sv(Edmonds et al., Mol. Biotech. 34:179-190 (2006)), CHO-S (Pichler etal., Biotechnol. Bioeng. 108:386-94 (2011)), dihydrofolate reductasenegative CHO cells (CHO/-DHFR, Urlaub and Chasin, 1980, Proc. Natl.Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Pat. No. 5,721,121);monkey kidney CV1 cells transformed by SV40 (COS cells, COS-7, ATCCCRL-1651); human embryonic kidney cells (e.g., 293 cells, or 293 cellssubcloned for growth in suspension culture, Graham et al., 1977, J. Gen.Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); monkeykidney cells (CV1, ATCC CCL-70); African green monkey kidney cells(VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse sertoli cells (TM4,Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells(HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lungcells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mousemammary tumor cells (MMT 060562, ATCC CCL-51); buffalo rat liver cells(BRL 3A, ATCC CRL-1442); TR1 cells (Mather, 1982, Ann. NY Acad. Sci.,383:44-68); MCR 5 cells; and FS4 cells.

For long-term, high-yield production of recombinant proteins, host cellscan be engineered to stably express a polypeptide (e.g., antibody). Hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements known in the art, including promoter, enhancer,sequences, transcription terminators, polyadenylation sites, andselectable markers. Methods commonly known in the art of recombinant DNAtechnology can be used to select a desired recombinant clone. In someembodiments, a cell is genetically engineered to increase level ofexpression of an endogenous polypeptide, e.g., by increasingtranscription of a gene encoding the polypeptide and/or increasing mRNAstability. In some embodiments, transcription of a gene encoding apolypeptide is increased by: altering the regulatory sequence of theendogenous gene, e.g., in a somatic cell, e.g., by the addition of apositive regulatory element, such as an enhancer or a DNA-binding sitefor a transcriptional activator; the deletion of a negative regulatoryelement, such as a DNA-binding site for a transcriptional repressor;and/or replacement of the endogenous regulatory sequence, or elementstherein, with that of another gene, thereby allowing the coding regionof the gene to be transcribed more efficiently.

Once a polypeptide described herein (e.g., an antibody described herein)has been produced by recombinant expression, it can be purified by anymethod known in the art for purification, for example, by chromatography(e.g., ion exchange, affinity, and sizing column chromatography),centrifugation, differential solubility, or by any other standardtechnique for the purification of proteins. For example, an antibody canbe isolated and purified by appropriately selecting and combiningaffinity columns such as Protein A column with chromatography columns,filtration, ultra filtration, salting-out and dialysis procedures (seeAntibodies: A Laboratory Manual, Ed Harlow, David Lane, Cold SpringHarbor Laboratory, 1988). Further, as described herein, a polypeptide(e.g., an antibody) can be fused to heterologous polypeptide sequencesto facilitate purification. Polypeptides having desired sugar chains canbe separated with a lectin column by methods known in the art (see,e.g., WO 02/30954).

In accordance with the present disclosure, there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are described inthe literature (see, e.g., Sambrook, Fritsch & Maniatis, MolecularCloning: A Laboratory Manual, Second Edition (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; DNA Cloning: A PracticalApproach, Volumes I and II (D. N. Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. (1985)); Transcription And Translation (B. D. Hames& S. J. Higgins, eds. (1984)); Animal Cell Culture (R. I. Freshney, ed.(1986)); Immobilized Cells and Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

Culture Methods

In general, polypeptides (e.g., antibodies) having target levels ofglycans (e.g., high mannose glycans, fucosylated glycans and/orsialylated glycans) can be produced by culturing cells in media thatcontains DMSO, e.g., a particular, effective level of DMSO (e.g., duringone or more stages of culture).

In some embodiments, cells genetically engineered to express apolypeptide are cultured (e.g., at one or more stages of culture) in amedium that includes DMSO, e.g., an elevated level of DMSO, to decreaselevels of G0F glycans and/or fucosylated glycans in a preparation of thepolypeptide expressed by the cells. In some embodiments, a level of G0Fglycans and/or fucosylated glycans is decreased relative to thecorresponding level(s) in a preparation of the polypeptide producedusing the same medium that does not include DMSO, e.g., an elevatedlevel of DMSO. In some embodiments, the decreased level of G0F glycansand/or fucosylated glycans is lower than the corresponding level(s) byat least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or100%, of the corresponding level.

In some embodiments, cells genetically engineered to express apolypeptide are cultured (e.g., at one or more stages of culture) in amedium that includes DMSO, e.g., an elevated level of DMSO, to increaselevels of high mannose glycans, afucosylated glycans, and/or sialylatedglycans in a preparation of the polypeptide expressed by the cells. Insome embodiments, a level of high mannose glycans, afucosylated glycans,and/or sialylated glycans is increased relative to the correspondinglevel(s) in a preparation of the polypeptide produced using the samemedium that does not include DMSO, e.g., an elevated level of DMSO. Insome embodiments, the increased level of high mannose glycans,afucosylated glycans, and/or sialylated glycans is higher than thecorresponding level(s) by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, ormore, of the corresponding level.

In some embodiments, cells genetically engineered to express apolypeptide are cultured (e.g., at one or more stages of culture) in amedium that includes a reduced level of DMSO, e.g., does not includeDMSO, to increase levels of G0F glycans and/or fucosylated glycans in apreparation of the polypeptide expressed by the cells. In someembodiments, a level of G0F glycans and/or fucosylated glycans isincreased relative to the corresponding level(s) in a preparation of thepolypeptide produced using the same medium that does not include areduced level of DMSO. In some embodiments, the increased level of G0Fglycans and/or fucosylated glycans is higher than the correspondinglevel(s) by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, or more, of thecorresponding level.

In some embodiments, cells genetically engineered to express apolypeptide are cultured (e.g., at one or more stages of culture) in amedium that includes a reduced level of DMSO, e.g., does not includeDMSO, to decrease levels of high mannose glycans, afucosylated glycans,and/or sialylated glycans in a preparation of the polypeptide expressedby the cells. In some embodiments, a level of high mannose glycans,afucosylated glycans, and/or sialylated glycans is decreased relative tothe corresponding level(s) in a preparation of the polypeptide producedusing the same medium that does not include a reduced level of DMSO. Insome embodiments, the decreased level of high mannose glycans,afucosylated glycans, and/or sialylated glycans is lower than thecorresponding level(s) by at least about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90%, 95%, or 100%, of the corresponding level.

As used herein, an “elevated level of DMSO” means a higher concentrationof DMSO than is present in a standard medium, a starting medium, amedium used at one or more stages of culture, and/or in a medium inwhich a polypeptide is produced. In some embodiments, DMSO is notpresent in a standard and/or starting medium, a medium used at one ormore other stages of culture, and/or in a medium in which a polypeptideis produced, and an “elevated level” is any amount of DMSO. A medium caninclude an elevated level of DMSO initially (i.e., at the start of aculture), and/or medium can be supplemented with DMSO to achieve anelevated level of DMSO at a particular time or times (e.g., at one ormore stages) during culturing.

In some embodiments, an elevated level of DMSO is an increase of atleast about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,200%, 250%, 300%, 350%, 400%, 450%, 500%, 550%, 600%, 650%, 700%, 750%,800%, 850%, 900%, 950%, 1000% or more, relative to a level of DMSO in astandard medium, a starting medium, a medium during one or more stagesof culture, and/or in a medium in which a polypeptide is produced.

In some embodiments, an elevated level of DMSO is an increase in level(v/v) of DMSO of at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%,1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%,3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%,4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, or more (v/v) DMSO,relative to a level (v/v) of DMSO in a standard medium, a startingmedium, a medium during one or more stages of culture, and/or in amedium in which a polypeptide is produced.

In some embodiments, an elevated level of DMSO is about 0.1%, 0.2%,0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%,1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%,2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%,3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, orhigher (v/v) DMSO. In some embodiments, an elevated level of DMSO isabout 0.1% to about 5% DMSO, e.g., about 0.1% to about 1% DMSO, about 1%to about 2% DMSO, about 2% to about 3% DMSO, about 3% to about 4% DMSO,about 4% to about 5% DMSO, about 0.1% to about 2.5% DMSO, about 2.5% toabout 5% DMSO, about 0.1% to about 2% DMSO, about 0.1% to about 3% DMSO,about 0.1% to about 4% DMSO, about 1% to about 5% DMSO, about 2% toabout 5% DMSO, or about 3% to about 5% DMSO.

As used herein, a “reduced level of DMSO” means a lower concentration ofDMSO than is present in a standard medium, a starting medium, a mediumused at one or more stages of culture, and/or in a medium in which apolypeptide is produced. A medium can include a reduced level of DMSOinitially (i.e., at the start of a culture), a medium can be diluted ata particular time or times (e.g., at one or more stages) duringculturing to reduce the level of DMSO, and/or a medium can be replacedwith a medium having a reduced level of DMSO at a particular time ortimes (e.g., at one or more stages) during culturing. In someembodiments, a reduced level of DMSO is 0% (v/v) DMSO or no detectablelevel of DMSO in a medium.

In some embodiments, a reduced level of DMSO is a decrease of at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, relative toa level of DMSO in a standard medium, a starting medium, a medium duringone or more stages of culture, and/or in a medium in which a polypeptideis produced.

In some embodiments, a reduced level of DMSO is a decrease in level(v/v) of DMSO of at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%,1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%,3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%,4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, or more, (v/v) DMSO,relative to a level (v/v) of DMSO in a standard medium, a startingmedium, a medium during one or more stages of culture, and/or in amedium in which a polypeptide is produced.

In some embodiments, a reduced level of DMSO is less than about 5%,4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%,3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%,2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%,1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or0.1%; or is 0% (v/v) DMSO.

Cells can be cultured in a variety of cell culture media known in theart, which are modified according to the disclosure to include DMSO asdescribed herein. Cell culture medium is understood by those of skill inthe art to refer to a nutrient solution in which cells, such as animalor mammalian cells, are grown. A cell culture medium generally includesone or more of the following components: an energy source (e.g., acarbohydrate such as glucose); amino acids; vitamins; lipids or freefatty acids; and trace elements, e.g., inorganic compounds or naturallyoccurring elements in the micromolar range. Cell culture medium can alsocontain additional components, such as hormones and other growth factors(e.g., insulin, transferrin, epidermal growth factor, serum, and thelike); salts (e.g., calcium, magnesium and phosphate); buffers (e.g.,HEPES); nucleosides and bases (e.g., adenosine, thymidine,hypoxanthine); antibiotics (e.g., gentamycin); and cell protectiveagents (e.g., a Pluronic polyol (Pluronic F68)).

In some embodiments, in addition to an elevated or reduced level ofDMSO, a cell culture medium includes lysine, cysteine, ammonium,manganese, copper, cobalt, putrescine, a peptone, glucose, galactose,galactosamine, glucosamine, glutamine, a lipid (e.g., cholesterol),and/or dextran sulfate. For example, a culture medium can include about0.1 g/L to about 30 g/L lysine; about 0.1 g/L to about 1 g/L cysteine;about 1 mM to about 50 mM ammonium; about 0.01 mM to about 0.5 mMmanganese; about 0.1 μM to about 0.5 mM copper; about 0.1 mg/L to about30 mg/L cobalt; about 0.01 mg/L to about 5 mg/L putrescine; about 0.1g/L to about 10 g/L glucose; about 0.5 g/L to about 30 g/L peptone,e.g., a non-animal derived peptone such as soy or cottonseed; about 1 μMto about 1 mM galactosamine; about 0.1 g/L to about 5 g/L glucosamine;and/or about 0.01 g/L to about 0.1 g/L dextran sulfate.

Media that has been prepared or commercially available can be modifiedaccording to the present disclosure for utilization in the methodsdescribed herein. Nonlimiting examples of such media include MinimalEssential Medium (MEM, Sigma, St. Louis, Mo.); Ham's F10 Medium (Sigma);Dulbecco's Modified Eagles Medium (DMEM, Sigma); RPM I-1640 Medium(Sigma); HyClone cell culture medium (HyClone, Logan, Utah); Power CHO2(Lonza Inc., Allendale, N.J.); and chemically-defined (CD) media, whichare formulated for particular cell types, e.g., CD-CHO Medium(Invitrogen, Carlsbad, Calif.). Culture medium suitable for particularcells being cultured can be determined by a person of ordinary skill inthe art without undue experimentation, and such medium can be alteredaccording to the disclosure.

Cell culture conditions (including pH, O₂, CO₂, agitation rate andtemperature) suitable for cellular production of polypeptides describedherein (e.g., antibodies) are those that are known in the art, such asconditions for batch, continuous, or fed-batch culturing of cells. Forexample, pH of cell culture medium is generally maintained at about 6.8to about 7.2.

In some embodiments, cells are cultured in one or more stages, and cellscan be cultured in medium having an elevated or reduced level of DMSO inone or more stages. For example, a culture method can include a firststage (e.g., using a medium having a reduced level of DMSO) and a secondstage (e.g., using a medium having an elevated level of DMSO). In someembodiments, a culture method can include a first stage (e.g., using amedium having an elevated level of DMSO) and a second stage (e.g., usinga medium having a reduced level of DMSO). In some embodiments, a culturemethod includes more than two stages, e.g., 3, 4, 5, 6, or more stages,and any stage can include medium having an elevated level of DMSO or areduced level of DMSO. The length of culture is not limiting. Forexample, a culture method can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more days. In some embodiments, a culture methodincludes at least two stages. For example, a first stage can includeculturing cells in medium having a reduced level of DMSO (e.g., forabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days), and a second stagecan include culturing cells in medium having an elevated level of DMSO(e.g., for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days).

In some embodiments, cells are cultured in an initial medium having areduced level of DMSO for 5 days, and medium having an elevated of DMSOis added to the culture of cells on day 6.

In some embodiments, a first stage of culture is a growth stage.Generally, during a growth stage, a cell culture undergoes a period ofexponential cell growth (the log phase) where cells are generallyrapidly dividing. In some embodiments, cells are cultured under suchconditions such that cell growth is maximized. The growth cycle andconditions for maximizing growth of host cells can be determined for aparticular host cell by a person of ordinary skill in the art withoutundue experimentation. In some embodiments, cells are maintained in agrowth stage for a period of between 1 and 10 days. In some embodiments,cells are cultured in a medium having a reduced level of DMSO or anelevated level of DMSO for all or part of a growth stage.

In some embodiments, a second stage of culture is a production stage.Generally, during a production stage, cell growth has plateaued or ismaintained at a near constant level. During a production stage,logarithmic cell growth has ended and polypeptide production isincreased. During this period of time, a medium can generally besupplemented to support continued polypeptide production and to achievea desired polypeptide product. In some embodiments, cells are maintainedin a production stage for a period of between 1 and 10 days. In someembodiments, cells are cultured in a medium having a reduced level ofDMSO or an elevated level of DMSO for all or part of a production stage.

In general, cell culture methods are classified as batch culture,continuous culture, and fed-batch culture. Any of these culture methodscan be used to grow cells that produce polypeptides (e.g., antibodies)having targeted levels of glycans (e.g., high mannose glycans,fucosylated glycans, and/or sialylated glycans).

Batch Culture

In batch culture, a small amount of seed culture solution is added to amedium and cells are grown without any addition of a new medium ordischarge of culture solution during culture. For the production ofpolypeptides (e.g., antibodies) having targeted levels of glycans (e.g.,high mannose glycans, fucosylated glycans, and/or sialylated glycans)using batch culture, the medium comprises an elevated level or a reducedlevel of DMSO at an initial stage of cell culture.

In some embodiments, polypeptides (e.g., antibodies) having targetedlevels of glycans (e.g., high mannose glycans, fucosylated glycans,and/or sialylated glycans) are produced by batch culture of cellsexpressing the polypeptide in a medium having an elevated level of DMSO.In some embodiments, cells are cultured in a medium having at leastabout 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%,1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%,2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%,3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%,4.8%, 4.9%, 5%, or higher (v/v) DMSO. In some embodiments, cells arecultured in a medium having at least about 0.1% to about 5% DMSO, e.g.,about 0.1% to about 1% DMSO, about 1% to about 2% DMSO, about 2% toabout 3% DMSO, about 3% to about 4% DMSO, about 4% to about 5% DMSO,about 0.1% to about 2.5% DMSO, about 2.5% to about 5% DMSO, about 0.1%to about 2% DMSO, about 0.1% to about 3% DMSO, about 0.1% to about 4%DMSO, about 1% to about 5% DMSO, about 2% to about 5% DMSO, or about 3%to about 5% DMSO.

In some embodiments, polypeptides (e.g., antibodies) having targetedlevels of glycans (e.g., high mannose glycans, fucosylated glycans,and/or sialylated glycans) are produced by batch culture of cellsexpressing the polypeptide in a medium having a reduced level of DMSO.In some embodiments, cells are cultured in a medium having less thanabout 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%,3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%,2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%,1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%,0.3%, 0.2%, or 0.1%; or having 0% (v/v) DMSO.

Continuous Culture

Continuous culture is a culture method in which a medium is added anddischarged continuously during culture. This continuous method includesperfusion culture. For example, in the production of polypeptides (e.g.,antibodies) having targeted levels of glycans (e.g., high mannoseglycans, fucosylated glycans, and/or sialylated glycans) usingcontinuous culture, level of DMSO in the medium can be adjusted at oneor more stages of culture to result in an elevated level or a reducedlevel of DMSO. In certain methods, culture medium used at a first stageof culture does not include an elevated level or a reduced level ofDMSO, but at a particular time point during continuous culture (such asduring all or part of a growth stage and/or a production stage), mediumadded during culture is elevated or reduced in the level of DMSO.

In some embodiments, polypeptides (e.g., antibodies) having targetedlevels of glycans (e.g., high mannose glycans, fucosylated glycans,and/or sialylated glycans) are produced by continuous culture of cellsexpressing the polypeptide in a medium having an elevated level of DMSO(e.g., during one or more stages of continuous culture). In someembodiments, cells are cultured, during one or more stages of continuousculture, in a medium having at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%,0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%,1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%,3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%,4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, or higher (v/v)DMSO. In some embodiments, cells are cultured, during one or more stagesof continuous culture, in a medium having at least about 0.1% to about5% DMSO, e.g., about 0.1% to about 1% DMSO, about 1% to about 2% DMSO,about 2% to about 3% DMSO, about 3% to about 4% DMSO, about 4% to about5% DMSO, about 0.1% to about 2.5% DMSO, about 2.5% to about 5% DMSO,about 0.1% to about 2% DMSO, about 0.1% to about 3% DMSO, about 0.1% toabout 4% DMSO, about 1% to about 5% DMSO, about 2% to about 5% DMSO, orabout 3% to about 5% DMSO.

In some embodiments, cells are cultured, during one or more stages ofcontinuous culture, in a medium having a reduced level of DMSO. In someembodiments, cells are cultured, during one or more stages of continuousculture, in a medium having less than about 5%, 4.9%, 4.8%, 4.7%, 4.6%,4.5%, 4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%,3.3%, 3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%,2.1%, 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%,0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%; or having 0%(v/v) DMSO.

Fed-Batch Culture

Fed-batch culture is a method between batch culture and continuousculture. In a fed-batch culture, a cell culture is fed or supplementedcontinuously or sequentially during culture, but unlike continuousculture, discharge of culture solution is not carried out duringculture. For example, for the production of polypeptides (e.g.,antibodies) having targeted levels of glycans (e.g., high mannoseglycans, fucosylated glycans, and/or sialylated glycans) using fed-batchculture, medium added during one or more stages of culture can have anelevated level or a reduced level of DMSO.

In some embodiments, polypeptides (e.g., antibodies) having targetedlevels of glycans (e.g., high mannose glycans, fucosylated glycans,and/or sialylated glycans) are produced by adding medium (at one or morestages) to a fed batch culture of cells expressing the polypeptidesufficient to achieve (at one or more stages) an elevated level of DMSO.In some embodiments, at least about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%,0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%,1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%,3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%,4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, or higher (v/v) DMSO inthe culture medium is achieved (e.g., by adding or supplementing withDMSO, e.g., at one or more stages). In some embodiments, a level of DMSOof at least about 0.1% to about 5% DMSO, e.g., about 0.1% to about 1%DMSO, about 1% to about 2% DMSO, about 2% to about 3% DMSO, about 3% toabout 4% DMSO, about 4% to about 5% DMSO, about 0.1% to about 2.5% DMSO,about 2.5% to about 5% DMSO, about 0.1% to about 2% DMSO, about 0.1% toabout 3% DMSO, about 0.1% to about 4% DMSO, about 1% to about 5% DMSO,about 2% to about 5% DMSO, or about 3% to about 5% DMSO, is achieved.

In some embodiments, polypeptides (e.g., antibodies) having targetedlevels of glycans (e.g., high mannose glycans, fucosylated glycans,and/or sialylated glycans) are produced by adding medium (at one or morestages) to a fed batch culture of cells expressing the polypeptidesufficient to achieve (at one or more stages) a reduced level of DMSO.In some embodiments, less than about 5%, 4.9%, 4.8%, 4.7%, 4.6%, 4.5%,4.4%, 4.3%, 4.2%, 4.1%, 4%, 3.9%, 3.8%, 3.7%, 3.6%, 3.5%, 3.4%, 3.3%,3.2%, 3.1%, 3%, 2.9%, 2.8%, 2.7%, 2.6%, 2.5%, 2.4%, 2.3%, 2.2%, 2.1%,2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%,0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%; or 0% (v/v) DMSO inthe culture medium is achieved (at one or more stages).

According to the present disclosure, cell culture can be carried outunder conditions for large or small scale production of polypeptides(e.g., antibodies), using culture vessels and/or culture apparatusesthat are conventionally employed for animal or mammalian cell culture.For example, tissue culture dishes, T-flasks, shaker flasks, and spinnerflasks can be used on a laboratory scale. For culturing on a largerscale (e.g., 1 L, 10 L, 100 L, 500 L, 5000 L, or more), a fluidized bedbioreactor, a hollow fiber bioreactor, a roller bottle culture, or astirred tank bioreactor system can be used (e.g., as described in U.S.Pat. Nos. 7,541,164 and 7,332,303).

In particular methods, levels of one or more glycans (e.g., high mannoseglycans, fucosylated glycans, and/or sialylated glycans) in apreparation of polypeptides (e.g., antibodies) are monitored during oneor more times (e.g., one or more stages) of cell culture, therebyallowing adjustment (e.g., increasing or decreasing the amount of DMSOin the culture) or possibly termination of the culture in order, forexample, to achieve a target level of polypeptides (e.g., antibodies)having targeted levels of glycans (e.g., high mannose glycans,fucosylated glycans, and/or sialylated glycans).

Polypeptides

Described herein are therapeutic preparations of polypeptides (e.g.,glycoproteins), and methods of making and using such preparations.Glycoproteins include, for example, any of a variety of hematologicagents (including, for instance, erythropoietin, blood-clotting factors,etc.), interferons, colony stimulating factors, antibodies, enzymes, andhormones. The identity of a particular glycoprotein is not intended tolimit the present disclosure, and a therapeutic preparation describedherein can include any glycoprotein of interest, e.g., a glycoproteinhaving an Fc region.

A glycoprotein described herein can include a target-binding domain thatbinds to a target of interest (e.g., binds to an antigen). For example,a glycoprotein, such as an antibody, can bind to a transmembranepolypeptide (e.g., receptor) or ligand (e.g., a growth factor).Exemplary molecular targets (e.g., antigens) for glycoproteins describedherein (e.g., antibodies) include CD proteins such as CD2, CD3, CD4,CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40, CD52; members ofthe ErbB receptor family such as the EGF receptor (EGFR, HER1, ErbB1),HER2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor; macrophagereceptors such as CRIg; tumor necrosis factors such as TNFα orTRAIL/Apo-2; cell adhesion molecules such as LFA-1, Mac1, p150,95,VLA-4, ICAM-1, VCAM and αvβ3 integrin including either α or β subunitsthereof (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growthfactors and receptors such as EGF, FGFR (e.g., FGFR3) and VEGF; IgE;cytokines such as IL1; cytokine receptors such as IL2 receptor; bloodgroup antigens; flk2/flt3 receptor; obesity (OB) receptor; mp1 receptor;CTLA-4; protein C; neutropilins; ephrins and receptors; netrins andreceptors; slit and receptors; chemokines and chemokine receptors suchas CCL5, CCR4, CCR5; amyloid beta; complement factors, such ascomplement factor D; lipoproteins, such as oxidized LDL (oxLDL);lymphotoxins, such as lymphotoxin alpha (LTa). Other molecular targetsinclude Tweak, B7RP-1, proprotein convertase subtilisin/kexin type 9(PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

Reference Polypeptides

In some embodiments, described herein are therapeutic polypeptide (e.g.,glycoprotein) having targeted levels of glycans (e.g., high mannoseglycans, fucosylated glycans, and/or sialylated glycans), where thetargeted levels are the levels of glycans (e.g., high mannose glycans,fucosylated glycans, and/or sialylated glycans) in a referencepolypeptide product (e.g., glycoprotein product). Nonlimiting, exemplaryreference glycoprotein products can include abatacept (Orencia®,Bristol-Myers Squibb), abciximab (ReoPro®, Roche), adalimumab (Humira®,Bristol-Myers Squibb), aflibercept (Eylea®, Regeneron Pharmaceuticals),alefacept (Amevive®, Astellas Pharma), alemtuzumab (Campath®,Genzyme/Bayer), basiliximab (Simulect®, Novartis), belatacept (Nulojix®,Bristol-Myers Squibb), belimumab (Benlysta®, GlaxoSmithKline),bevacizumab (Avastin®, Roche), canakinumab (Ilaris®, Novartis),brentuximab vedotin (Adcetris®, Seattle Genetics), certolizumab(CIMZIA®, UCB, Brussels, Belgium), cetuximab (Erbitux®, Merck-Serono),daclizumab (Zenapax®, Hoffmann-La Roche), denileukin diftitox (Ontak®,Eisai), denosumab (Prolia®, Amgen; Xgeva®, Amgen), eculizumab (Soliris®,Alexion Pharmaceuticals), efalizumab (Raptiva®, Genentech), etanercept(Enbrel®, Amgen-Pfizer), gemtuzumab (Mylotarg®, Pfizer), golimumab(Simponi®, Janssen), ibritumomab (Zevalin®, Spectrum Pharmaceuticals),infliximab (Remicade®, Centocor), ipilimumab (Yervoy™ Bristol-MyersSquibb), muromonab (Orthoclone OKT3®, Janssen-Cilag), natalizumab(Tysabri®, Biogen Idec, Elan), ofatumumab (Arzerra®, GlaxoSmithKline),omalizumab (Xolair®, Novartis), palivizumab (Synagis®, MedImmune),panitumumab (Vectibix®, Amgen), ranibizumab (Lucentis®, Genentech),rilonacept (Arcalyst®, Regeneron Pharmaceuticals), rituximab (MabThera®,Roche), tocilizumab (Actemra®, Genentech; RoActemra, Hoffman-La Roche)tositumomab (Bexxar®, GlaxoSmithKline), and trastuzumab (Herceptin®,Roche).

In some embodiments, a level of one or more glycans (e.g., high mannoseglycans, fucosylated glycans, and/or sialylated glycans) in a referencepolypeptide product is determined by analyzing one or more preparations(e.g., one or more lots) of the reference polypeptide. In someembodiments, a level of one or more glycans (e.g., high mannose glycans,fucosylated glycans, and/or sialylated glycans) in a referencepolypeptide product is a range of the one or more glycans in two or morepreparations of the reference polypeptide (e.g., two or more lots of thereference polypeptide product). In some embodiments, a level of one ormore glycans is a range (e.g., spanning a lowest level of the one ormore glycans to a highest level of the one or more glycans) in two ormore lots of the reference polypeptide product.

N-Linked Glycosylation

N-linked oligosaccharide chains are added to a protein in the lumen ofthe endoplasmic reticulum (see Molecular Biology of the Cell, GarlandPublishing, Inc. (Alberts et al., 1994)). Specifically, an initialoligosaccharide (typically 14-sugar) is added to the amino group on theside chain of an asparagine residue contained within the targetconsensus sequence of Asn-X-Ser/Thr, where X may be any amino acidexcept proline. The structure of this initial oligosaccharide is commonto most eukaryotes, and contains 3 glucose, 9 mannose, and 2N-acetylglucosamine residues. This initial oligosaccharide chain can betrimmed by specific glycosidase enzymes in the endoplasmic reticulum,resulting in a short, branched core oligosaccharide composed of twoN-acetylglucosamine and three mannose residues.

N-glycans can be subdivided into three distinct groups called “highmannose type”, “hybrid type”, and “complex type”, with a commonpentasaccharide core (Man(alpha1,6)-(Man(alpha1,3))-Man(beta1,4)-GlcpNAc(beta 1,4)-GlcpNAc(beta1,N)-Asn) occurring in all three groups.

After initial processing in the endoplasmic reticulum, the glycoproteinis transported to the Golgi where further processing may take place. Ifthe glycan is transferred to the Golgi before it is completely trimmedto the core pentasaccharide structure, it results in a “high-mannoseglycan”.

Additionally or alternatively, one or more monosaccharides units ofN-acetylglucosamine may be added to core mannose subunits to form a“complex glycan”. Galactose may be added to N-acetylglucosaminesubunits, and sialic acid subunits may be added to galactose subunits,resulting in chains that terminate with any of a sialic acid, agalactose or an N-acetylglucosamine residue. Additionally, a fucoseresidue may be added to an N-acetylglucosamine residue of the coreoligosaccharide. Each of these additions is catalyzed by specificglycosyl transferases, known in the art.

Sialic acids are a family of 9-carbon monosaccharides with heterocyclicring structures. They bear a negative charge via a carboxylic acid groupattached to the ring as well as other chemical decorations includingN-acetyl and N-glycolyl groups. The two main types of sialyl residuesfound in glycoproteins produced in mammalian expression systems areN-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc).These usually occur as terminal structures attached to galactose (Gal)residues at the non-reducing termini of both N- and O-linked glycans.The glycosidic linkage configurations for these sialyl groups can beeither α2,3 or α2,6.

“Hybrid glycans” comprise characteristics of both high-mannose andcomplex glycans. For example, one branch of a hybrid glycan may compriseprimarily or exclusively mannose residues, while another branch maycomprise N-acetylglucosamine, sialic acid, and/or galactose sugars.

N-Linked Glycosylation in Antibodies

Antibodies are glycosylated at conserved, N-linked glycosylation sitesin the Fc regions of immunoglobulin heavy chains. For example, eachheavy chain of an IgG antibody has a single N-linked glycosylation siteat Asn297 of the CH2 domain (see Jefferis, Nature Reviews 8:226-234(2009)). IgA antibodies have N-linked glycosylation sites within the CH2and CH3 domains, IgE antibodies have N-linked glycosylation sites withinthe CH3 domain, and IgM antibodies have N-linked glycosylation siteswithin the CH1, CH2, CH3, and CH4 domains (see Arnold et al., J. Biol.Chem. 280:29080-29087 (2005); Mattu et al., J. Biol. Chem. 273:2260-2272(1998); Nettleton et al., Int. Arch. Allergy Immunol. 107:328-329(1995)).

Each antibody isotype has a distinct variety of N-linked carbohydratestructures in the constant regions. For example, IgG has a singleN-linked biantennary carbohydrate at Asn297 of the CH2 domain in each Fcpolypeptide of the Fc region, which also contains the binding sites forC1q and FcγR (see Jefferis et al., Immunol. Rev. 163:59-76 (1998); andWright et al., Trends Biotech 15:26-32 (1997)). For human IgG, the coreoligosaccharide normally consists of GlcNAc₂Man₃GlcNAc, with differingnumbers of outer residues. Variation among individual IgG can occur viaattachment of galactose and/or galactose-sialic acid at one or bothterminal GlcNAc or via attachment of a third GlcNAc arm (bisectingGlcNAc), and/or attachment of fucose.

Antibodies

The basic structure of an IgG antibody is illustrated in FIG. 1. Asshown in FIG. 1, an IgG antibody consists of two identical lightpolypeptide chains and two identical heavy polypeptide chains linkedtogether by disulphide bonds. The first domain located at the aminoterminus of each chain is variable in amino acid sequence, providingantibody binding specificities found in each individual antibody. Theseare known as variable heavy (VH) and variable light (VL) regions. Theother domains of each chain are relatively invariant in amino acidsequence and are known as constant heavy (CH) and constant light (CL)regions. As shown in FIG. 1, for an IgG antibody, the light chainincludes one variable region (VL) and one constant region (CL). An IgGheavy chain includes a variable region (VH), a first constant region(CH1), a hinge region, a second constant region (CH2), and a thirdconstant region (CH3). In IgE and IgM antibodies, the heavy chainincludes an additional constant region (CH4).

Antibodies described herein can include, for example, monoclonalantibodies, polyclonal antibodies (e.g., WIG), multispecific antibodies,human antibodies, humanized antibodies, camelized antibodies, chimericantibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), andanti-idiotypic (anti-Id) antibodies, and antigen-binding fragments ofany of the above. Antibodies can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclass.

The term “Fc fragment”, as used herein, refers to one or more fragmentsof an Fc region that retains an Fc function and/or activity describedherein, such as binding to an Fc receptor. Examples of such fragmentsinclude fragments that include an N-linked glycosylation site of an Fcregion (e.g., an Asn297 of an IgG heavy chain or homologous sites ofother antibody isotypes), such as a CH2 domain. The term “antigenbinding fragment” of an antibody, as used herein, refers to one or morefragments of an antibody that retain the ability to specifically bind toan antigen. Examples of binding fragments encompassed within the term“antigen binding fragment” of an antibody include a Fab fragment, aF(ab′)₂ fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAbfragment (Ward et al., (1989) Nature 341:544-546), and an isolatedcomplementarity determining region (CDR). These antibody fragments canbe obtained using conventional techniques known to those with skill inthe art, and fragments can be screened for utility in the same manner asare intact antibodies.

Reference glycoproteins described herein (e.g., reference antibodies) orfragments thereof can be produced by any method known in the art forsynthesizing glycoproteins (e.g., antibodies) (see, e.g., Harlow et al.,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO92/22324; WO 98/46645). Chimeric antibodies can be produced usingmethods described in, e.g., Morrison, 1985, Science 229:1202, andhumanized antibodies by methods described in, e.g., U.S. Pat. No.6,180,370.

Additional reference antibodies described herein are bispecificantibodies and multivalent antibodies, as described in, e.g., Segal etal., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol.147: 60 (1991).

Glycoprotein Conjugates

The disclosure includes glycoproteins (or Fc regions or Fc fragmentscontaining one or more N-glycosylation sites thereof) that areconjugated or fused to one or more heterologous moieties. Heterologousmoieties include, but are not limited to, peptides, polypeptides,proteins, fusion proteins, nucleic acid molecules, small molecules,mimetic agents, synthetic drugs, inorganic molecules, and organicmolecules. In some instances, a glycoprotein conjugate is a fusionprotein that comprises a peptide, polypeptide, protein scaffold, scFv,dsFv, diabody, Tandab, or an antibody mimetic fused to an Fc region,such as a glycosylated Fc region. A fusion protein can include a linkerregion connecting an Fc region to a heterologous moiety (see, e.g.,Hallewell et al. (1989), J. Biol. Chem. 264, 5260-5268; Alfthan et al.(1995), Protein Eng. 8, 725-731; Robinson & Sauer (1996)).

Exemplary, nonlimiting reference glycoprotein conjugate products includeabatacept (Orencia®, Bristol-Myers Squibb), aflibercept (Eylea®,Regeneron Pharmaceuticals), alefacept (Amevive®, Astellas Pharma),belatacept (Nulojix®, Bristol-Myers Squibb), denileukin diftitox(Ontak®, Eisai), etanercept (Enbrel®, Amgen-Pfizer), and rilonacept(Arcalyst®, Regeneron Pharmaceuticals).

In some instances, a glycoprotein conjugate includes an Fc region (or anFc fragment containing one or more N-glycosylations site thereof)conjugated to a heterologous polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids.

In some instances, a glycoprotein conjugate includes an Fc region (or anFc fragment containing one or more N-glycosylation sites thereof)conjugated to one or more marker sequences, such as a peptide tofacilitate purification. A particular marker amino acid sequence is ahexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). Otherpeptide tags useful for purification include, but are not limited to,the hemagglutinin “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767)and the “Flag” tag.

In other instances, a glycoprotein conjugate includes an Fc region (orFc fragment containing one or more N-glycosylation sites thereof)conjugated to a diagnostic or detectable agent. Such fusion proteins canbe useful for monitoring or prognosing development or progression ofdisease or disorder as part of a clinical testing procedure, such asdetermining efficacy of a particular therapy. Such diagnosis anddetection can be accomplished by coupling a glycoprotein to detectablesubstances including, but not limited to, various enzymes, such as butnot limited to horseradish peroxidase, alkaline phosphatase,beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as,but not limited to, streptavidin/biotin and avidin/biotin; fluorescentmaterials, such as, but not limited to, umbelliferone, fluorescein,fluorescein isothiocynate, rhodamine, dichlorotriazinylaminefluorescein, dansyl chloride or phycoerythrin; luminescent materials,such as, but not limited to, luminol; bioluminescent materials, such asbut not limited to, luciferase, luciferin, and aequorin; radioactivematerials, such as but not limited to iodine (¹³¹I, ¹²⁵I, ¹²³I ) carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In),technetium (⁹⁹Tc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium(¹⁶³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu,¹⁵³Gd, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re,¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ⁵¹Cr, ⁵⁴Mn,⁷⁵Se, ¹¹³Sn, and ¹¹⁷Sn; positron emitting metals using various positronemission tomographies, non-radioactive paramagnetic metal ions, andmolecules that are radiolabelled or conjugated to specificradioisotopes.

Techniques for conjugating therapeutic moieties to antibodies are wellknown (see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56. (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987)).

Glycan Evaluation

In some embodiments, glycans of glycoproteins are analyzed by anyavailable suitable method. In some instances, glycan structure andcomposition as described herein are analyzed, for example, by one ormore, enzymatic, chromatographic, mass spectrometry (MS),chromatographic followed by MS, electrophoretic methods, electrophoreticmethods followed by MS, nuclear magnetic resonance (NMR) methods, andcombinations thereof. Exemplary enzymatic methods include contacting aglycoprotein preparation with one or more enzymes under conditions andfor a time sufficient to release one or more glycan(s) (e.g., one ormore exposed glycan(s)). In some instances, the one or more enzymesinclude(s) PNGase F. Exemplary chromatographic methods include, but arenot limited to, Strong Anion Exchange chromatography using PulsedAmperometric Detection (SAX-PAD), liquid chromatography (LC), highperformance liquid chromatography (HPLC), ultra performance liquidchromatography (UPLC), thin layer chromatography (TLC), amide columnchromatography, and combinations thereof. Exemplary mass spectrometry(MS) include, but are not limited to, tandem MS, LC-MS, LC-MS/MS, matrixassisted laser desorption ionisation mass spectrometry (MALDI-MS),Fourier transform mass spectrometry (FTMS), ion mobility separation withmass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), andcombinations thereof. Exemplary electrophoretic methods include, but arenot limited to, capillary electrophoresis (CE), CE-MS, gelelectrophoresis, agarose gel electrophoresis, acrylamide gelelectrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE)followed by Western blotting using antibodies that recognize specificglycan structures, and combinations thereof. Exemplary nuclear magneticresonance (NMR) include, but are not limited to, one-dimensional NMR(1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopymagnetic-angle spinning NMR (COSY-NMR), total correlated spectroscopyNMR (TOCSY-NMR), heteronuclear single-quantum coherence NMR (HSQC-NMR),heteronuclear multiple quantum coherence (HMQC-NMR), rotational nuclearoverhauser effect spectroscopy NMR (ROESY-NMR), nuclear overhausereffect spectroscopy (NOESY-NMR), and combinations thereof.

In some instances, techniques described herein may be combined with oneor more other technologies for the detection, analysis, and or isolationof glycans or glycoproteins. For example, in certain instances, glycansare analyzed in accordance with the present disclosure using one or moreavailable methods (to give but a few examples, see Anumula, Anal.Biochem., 350(1):1, 2006; Klein et al., Anal. Biochem., 179:162, 1989;and/or Townsend, R.R. Carbohydrate Analysis” High Performance LiquidChromatography and Capillary Electrophoresis., Ed. Z. El Rassi, pp181-209, 1995; WO2008/128216; WO2008/128220; WO2008/128218;WO2008/130926; WO2008/128225; WO2008/130924; WO2008/128221;WO2008/128228; WO2008/128227; WO2008/128230; WO2008/128219;WO2008/128222; WO2010/071817; WO2010/071824; WO2010/085251;WO2011/069056; and WO2011/127322, each of which is incorporated hereinby reference in its entirety). For example, in some instances, glycansare characterized using one or more of chromatographic methods,electrophoretic methods, nuclear magnetic resonance methods, andcombinations thereof. In some embodiments, glycans are analyzed bylabeling with a fluorescent dye and measuring levels of fluorescence.

In some instances, methods for evaluating one or more target proteinspecific parameters, e.g., in a glycoprotein preparation, e.g., one ormore of the parameters disclosed herein, can be performed by one or moreof the methods listed in Table 1.

TABLE 1 Exemplary methods of evaluating parameters: Method(s) Relevantliterature Parameter C18 UPLC Mass Spec.* Chen and Flynn, Anal.Biochem., Glycan(s) 370:147-161 (2007) (e.g., N-linked glycan, exposedChen and Flynn, J. Am. Soc. Mass N-linked glycan, glycan Spectrom.,20:1821-1833 (2009) detection, glycan identification, andcharacterization; site specific glycation; glycoform detection (e.g.,parameters 1-7); percent glycosylation; and/or aglycosyl) Bioanalyzer(reducing/ Forrer et al., Anal. Biochem., Glycan (e.g., N-linked glycan,non-reducing)* 334:81-88 (2004) exposed N-linked glycan) (including, forexample, glycan detection, identification, and characterization; sitespecific glycation; glycoform detection; percent glycosylation; and/oraglycosyl) LC-MS (reducing/non- Dick et al., Biotechnol. Bioeng., Glycan(e.g., N-linked glycan, reducing/alkylated)* 100:1132-1143 (2008)exposed N-linked glycan) Goetze et al., Glycobiol., 21:949-959(including, for example, glycan (2011) detection, identification, andXie et al., mAbs, 2:379-394 (2010) characterization; site specificglycation; glycoform detection; percent glycosylation; and/or aglycosyl)Anion-exchange Ahn et al., J. Chrom. B, 878:403-408 Sialylated glycanchromatography (2010) 1,2-diamino-4,5- Hokke et al., FEBS Lett.,275:9-14 Sialic acid methylenedioxybenzene (1990) (DMB) labeling method*Methods include removal (e.g., enzymatic, chemical, and physical) ofglycansThe literature recited above are hereby incorporated by reference intheir entirety or, in the alternative, to the extent that they pertainto one or more of the methods for determining a parameter describedherein.

Pharmaceutical Compositions and Administration

A glycoprotein described herein can be incorporated (e.g., formulated)into a pharmaceutical composition. Such a pharmaceutical composition isuseful as an alternative and/or improved composition for the preventionand/or treatment of one or more diseases relative to a correspondingreference glycoprotein. Pharmaceutical compositions comprising aglycoprotein can be formulated by methods known to those skilled in theart. The pharmaceutical composition can be administered parenterally inthe form of an injectable formulation comprising a sterile solution orsuspension in water or another pharmaceutically acceptable liquid. Forexample, the pharmaceutical composition can be formulated by suitablycombining the glycoprotein with pharmaceutically acceptable vehicles ormedia, such as sterile water and physiological saline, vegetable oil,emulsifier, suspension agent, surfactant, stabilizer, flavoringexcipient, diluent, vehicle, preservative, binder, followed by mixing ina unit dose form required for generally accepted pharmaceuticalpractices. The amount of active ingredient included in thepharmaceutical preparations is such that a suitable dose within thedesignated range is provided.

A sterile composition for injection can be formulated in accordance withconventional pharmaceutical practices using distilled water forinjection as a vehicle. For example, physiological saline or an isotonicsolution containing glucose and other supplements such as D-sorbitol,D-mannose, D-mannitol, and sodium chloride may be used as an aqueoussolution for injection, optionally in combination with a suitablesolubilizing agent, for example, alcohol such as ethanol and polyalcoholsuch as propylene glycol or polyethylene glycol, and a nonionicsurfactant such as polysorbate ⁸⁰™, HCO-50 and the like.

Nonlimiting examples of oily liquid include sesame oil and soybean oil,and it may be combined with benzyl benzoate or benzyl alcohol as asolubilizing agent. Other items that may be included are a buffer suchas a phosphate buffer, or sodium acetate buffer, a soothing agent suchas procaine hydrochloride, a stabilizer such as benzyl alcohol orphenol, and an antioxidant. A formulated injection can be packaged in asuitable ampule.

In some instances, a level of one or more glycans described herein canbe compared to a predetermined level (e.g., a corresponding level in areference standard), e.g., to make a decision regarding the compositionof the polypeptide preparation, e.g., a decision to classify, select,accept or discard, release or withhold, process into a drug product,ship, move to a different location, formulate, label, package, releaseinto commerce, or sell or offer for sale the polypeptide, e.g., arecombinant antibody. In other instances, the decision can be to accept,modify or reject a production parameter or parameters used to make thepolypeptide, e.g., an antibody. Particular, nonlimiting examples ofreference standards include a control level (e.g., a polypeptideproduced by a different method) or a range or value in a productspecification (e.g., an FDA label or Physician's Insert) or qualitycriterion for a pharmaceutical preparation containing the polypeptidepreparation.

In some instances, methods (i.e., evaluation, identification, andproduction methods) include taking action (e.g., physical action) inresponse to the methods disclosed herein. For example, a polypeptidepreparation is classified, selected, accepted or discarded, released orwithheld, processed into a drug product, shipped, moved to a differentlocation, formulated, labeled, packaged, released into commerce, or soldor offered for sale, depending on whether the preselected or targetvalue is met. In some instances, processing may include formulating(e.g., combining with pharmaceutical excipients), packaging (e.g., in asyringe or vial), labeling, or shipping at least a portion of thepolypeptide preparation. In some instances, processing includesformulating (e.g., combining with pharmaceutical excipients), packaging(e.g., in a syringe or vial), and labeling at least a portion of thepreparation as a drug product described herein. Processing can includedirecting and/or contracting another party to process as describedherein.

In some instances, a biological activity of a polypeptide preparation(e.g., an antibody preparation) is assessed. Biological activity of thepreparation can be analyzed by any known method. In some embodiments, abinding activity of a polypeptide is assessed (e.g., binding to areceptor). In some embodiments, a therapeutic activity of a polypeptideis assessed (e.g., an activity of a polypeptide in decreasing severityor symptom of a disease or condition, or in delaying appearance of asymptom of a disease or condition). In some embodiments, a pharmacologicactivity of a polypeptide is assessed (e.g., bioavailability,pharmacokinetics, pharmacodynamics). For methods of analyzingbioavailability, pharmacokinetics, and pharmacodynamics of glycoproteintherapeutics, see, e.g., Weiner et al., J. Pharm. Biomed. Anal.15(5):571-9, 1997; Srinivas et al., J. Pharm. Sci. 85(1):1-4, 1996; andSrinivas et al., Pharm. Res. 14(7):911-6, 1997.

The particular biological activity or therapeutic activity that can betested will vary depending on the particular polypeptide (e.g.,antibody). The potential adverse activity or toxicity (e.g., propensityto cause hypertension, allergic reactions, thrombotic events, seizures,or other adverse events) of polypeptide preparations can be analyzed byany available method. In some embodiments, immunogenicity of apolypeptide preparation is assessed, e.g., by determining whether thepreparation elicits an antibody response in a subject.

Route of administration can be parenteral, for example, administrationby injection, transnasal administration, transpulmonary administration,or transcutaneous administration. Administration can be systemic orlocal by intravenous injection, intramuscular injection, intraperitonealinjection, subcutaneous injection.

A suitable means of administration can be selected based on the age andcondition of the patient. A single dose of the pharmaceuticalcomposition containing a modified glycoprotein can be selected from arange of 0.001 to 1000 mg/kg of body weight. On the other hand, a dosecan be selected in the range of 0.001 to 100000 mg/body weight, but thepresent disclosure is not limited to such ranges. The dose and method ofadministration varies depending on the weight, age, condition, and thelike of the patient, and can be suitably selected as needed by thoseskilled in the art.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. Unless otherwise defined, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which this inventionbelongs. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described herein.

The disclosure is further illustrated by the following example. Theexample is provided for illustrative purposes only. It is not to beconstrued as limiting the scope or content of the disclosure in any way.

Example Effect of DMSO on Glycosylation Methods

The effect of DMSO on antibody glycoforms of a model antibody producedby CHO cells was analyzed. CHO cells were grown initially in base media(Power CHO2, Catalog # BE15-771, Lonza Inc., Allendale, N.J.) containingsoy hydrolysate, Lonza Power Feed A and additional supplements. On Day3, the cells were fed with Lonza Power Feed A and soy hydrolysate. OnDay 6, the cells were fed with Lonza Power Feed A medium and additionalsupplements with 2.5% DMSO (final concentration) or without DMSO. Cellswere fed 2 g/L glucose when the glucose concentration dropped below 2g/L. Cells were harvested on Days 10, 12, or 14, and the antibodiesproduced were evaluated for titer and glycan composition.

Relative quantitation of each glycoform was performed on glycansenzymatically released from the antibody using PNGase F and labeled withthe fluorophore 2-aminobenzamide. The relative quantities were based onthe fluorescence intensity of the relevant species relative to the totalfluorescence intensity in the chromatogram. All glycoforms eluting atthe same retention time were considered together.

Results

The presence or absence of DMSO in the culture medium had minimal impacton titer or maximum VCD. Cells grown in the presence of DMSO had anaverage maximum VCD of 8.0×10⁶ cells/mL and a titer of 1.02 g/L, whereascells grown in the absence of DMSO had an average maximum VCD of 8.1×10⁶cells/mL and a titer of 0.92 g/L.

As shown in FIG. 2A, the level of G0F was decreased in antibodies grownin the presence of DMSO (about 7% decrease on day 14). Surprisingly, asshown in FIG. 2B, the levels of G1F and G2F were only slightly impactedby the presence of DMSO (by about 1% or less). This indicates that thepresence of DMSO did not shift the G0F/G1F/G2F distribution by alteringgalactosylation levels. Rather, this finding suggests that G0F wasdiverted to other species. A concomitant increase was seen in 3,3,1,0,0glycans (structure shown in FIG. 6; data not shown). As 3,3,1,0,0 isconverted to G0F by GnTII, without wishing to be bound by theory, DMSOmay be acting, at least in part, by altering the conversion of 3,3,1,0,0to G0F by GnTII. Further, as shown in FIG. 2A, the presence of DMSO hada temporal effect on G0F levels, which declined over days 10, 12, and14.

The impact of DMSO on high mannose glycans is shown in FIGS. 3A and 3B.As shown in FIG. 3A, DMSO increased the total level of high mannoseglycans compared to no DMSO. Further, DMSO led to increased levels ofhigh mannose 6, high mannose 3, and high mannose 5 glycans (see FIG.3B).

DMSO also affected the level of fucosylated glycans, resulting in adecreased level of fucosylated glycans (FIG. 4A), with an increase inafucosylated glycans (FIG. 4B). Further, DMSO resulted in an increase insialylated glycans (FIG. 5A), with a specific increase in the level of3,5,1,1,0 sialylated glycan (FIG. 5B; structure shown in FIG. 6).

This Example demonstrates that culturing cells in DMSO can be used toproduce polypeptides, expressed by the cells, having particular levelsof glycans.

EQUIVALENTS

It is to be understood that while the disclosure has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1-31. (canceled)
 32. A method of producing a recombinant proteinpreparation having a target value of high mannose glycans, the methodcomprising: (a) providing a mammalian cell genetically engineered toexpress a recombinant protein; (b) culturing the cell in a culturemedium comprising dimethylsulfoxide (DMSO) under conditions in which thecell expresses the recombinant protein; and (c) harvesting a preparationof the recombinant protein produced by the cell that meets the targetvalue, wherein the target value is 0.1% to 20% high mannose glycans. 33.A method of producing a recombinant protein preparation, the methodcomprising: (a) providing a mammalian cell genetically engineered toexpress a recombinant protein; (b) culturing the cell in a culturemedium comprising DMSO under conditions in which the cell expresses therecombinant protein; (c) harvesting a preparation of the recombinantprotein produced by the cell; and (d) formulating the preparation into adrug product if the preparation comprises 0.1% to 20% high mannoseglycans.
 34. The method of claim 32 or 33, wherein the mammalian cell isa Chinese Hamster Ovary (CHO) cell.
 35. The method of claim 32 or 33,wherein the CHO cell is a CHO-K1 or CHO-DG44 cell.
 36. The method ofclaim 32 or 33, wherein the mammalian cell is a murine myeloma cell. 37.The method of claim 36, wherein the murine myeloma cell is an NS0 orSp2/0 cell.
 38. The method of claim 32 or 33, wherein the culture mediumcomprises 0.5% to 5% DMSO.
 39. The method of claim 32 or 33, wherein theculture medium comprises 1% to 2% DMSO.
 40. The method of claim 32 or33, wherein the culture medium comprises 2% to 3% DMSO.
 41. The methodof claim 32 or 33, wherein the culture medium comprises 3% to 4% DMSO.42. The method of claim 32 or 33, wherein the culture medium comprises2% to 5% DMSO.
 43. The method of claim 32, wherein the target value is5% to 10% high mannose glycans.
 44. The method of claim 32, wherein thetarget value is 10% to 20% high mannose glycans.
 45. The method of claim33, wherein the preparation is formulated into a drug product if thepreparation comprises 5% to 10% high mannose glycans.
 46. The method ofclaim 33, wherein the preparation is formulated into a drug product ifthe preparation comprises 10% to 20% high mannose glycans.
 47. Themethod of claim 33, wherein the recombinant protein is abatacept. 48.The method of claim 33, wherein the recombinant protein is abciximab.49. The method of claim 33, wherein the recombinant protein isadalimumab.
 50. The method of claim 33, wherein the recombinant proteinis aflibercept.
 51. The method of claim 33, wherein the recombinantprotein is alefacept.
 52. The method of claim 33, wherein therecombinant protein is alemtuzumab.
 53. The method of claim 33, whereinthe recombinant protein is basiliximab.
 54. The method of claim 33,wherein the recombinant protein is bevacizumab.
 55. The method of claim33, wherein the recombinant protein is belatacept.
 56. The method ofclaim 33, wherein the recombinant protein is certolizumab.
 57. Themethod of claim 33, wherein the recombinant protein is cetuximab. 58.The method of claim 33, wherein the recombinant protein is daclizumab.59. The method of claim 33, wherein the recombinant protein iseculizumab.
 60. The method of claim 33, wherein the recombinant proteinis efalizumab.
 61. The method of claim 33, wherein the recombinantprotein is entanercept.
 62. The method of claim 33, wherein therecombinant protein is gemtuzumab.
 63. The method of claim 33, whereinthe recombinant protein is ibritumomab.
 64. The method of claim 33,wherein the recombinant protein is infliximab.
 65. The method of claim33, wherein the recombinant protein is muromonab-CD3.
 66. The method ofclaim 33, wherein the recombinant protein is natalizumab.
 67. The methodof claim 33, wherein the recombinant protein is omalizumab.
 68. Themethod of claim 33, wherein the recombinant protein is palivizumab. 69.The method of claim 33, wherein the recombinant protein is panitumumab.70. The method of claim 33, wherein the recombinant protein isranibizumab.
 71. The method of claim 33, wherein the recombinant proteinis rilonacept.
 72. The method of claim 33, wherein the recombinantprotein is rituximab.
 73. The method of claim 33, wherein therecombinant protein is tositumomab.
 74. The method of claim 33, whereinthe recombinant protein is trastuzumab.
 75. The method of claim 32 or33, further comprising evaluating a level of high mannose glycans in therecombinant protein preparation.
 76. A method of producing an adalimumabpreparation having a target value of high mannose glycans, the methodcomprising: (a) providing a CHO cell genetically engineered to expressadalimumab; (b) culturing the CHO cell in a culture medium comprising0.5% to 5% DMSO under conditions in which the CHO cell expressesadalimumab; and (c) harvesting a preparation of adalimumab produced bythe CHO cell that meets the target value, wherein the target value is alevel of high mannose glycans at least 20% higher than a level of highmannose glycans in an adalimumab preparation produced by culturing theCHO cell in the medium not comprising DMSO.
 77. The method of claim 76,wherein the target value is a level of high mannose glycans at least 30%higher than a level of high mannose glycans in an adalimumab preparationproduced by culturing the cell in the medium not comprising DMSO. 78.The method of claim 76, wherein the target value is a level of highmannose glycans at least 50% higher than a level of high mannose glycansin an adalimumab preparation produced by culturing the cell in themedium not comprising DMSO.
 79. The method of claim 76, wherein thetarget value is a level of high mannose glycans at least 70% higher thana level of high mannose glycans in an adalimumab preparation produced byculturing the cell in the medium not comprising DMSO.
 80. The method ofclaim 76, wherein the target value is a level of high mannose glycans atleast 100% higher than a level of high mannose glycans in an adalimumabpreparation produced by culturing the cell in the medium not comprisingDMSO.
 81. A method of producing an adalimumab preparation, the methodcomprising: (a) providing a CHO cell genetically engineered to expressadalimumab; (b) culturing the CHO cell in a culture medium comprising0.5% to 5% DMSO under conditions in which the CHO cell expressesadalimumab; (c) harvesting a preparation of the adalimumab produced bythe CHO cell; and (d) formulating the adalimumab preparation into a drugproduct if the adalimumab preparation comprises a level of high mannoseglycans at least 20% higher than a level of high mannose glycans in anadalimumab preparation produced by culturing the CHO cell in the mediumnot comprising DMSO.
 82. The method of claim 81, wherein the adalimumabpreparation is formulated into a drug product if the adalimumabpreparation comprises a level of high mannose glycans at least 30%higher than a level of high mannose glycans in an adalimumab preparationproduced by culturing the cell in the medium not comprising DMSO. 83.The method of claim 81, wherein the adalimumab preparation is formulatedinto a drug product if the adalimumab preparation comprises a level ofhigh mannose glycans at least 50% higher than a level of high mannoseglycans in an adalimumab preparation produced by culturing the cell inthe medium not comprising DMSO.
 84. The method of claim 81, wherein theadalimumab preparation is formulated into a drug product if theadalimumab preparation comprises a level of high mannose glycans atleast 70% higher than a level of high mannose glycans in an adalimumabpreparation produced by culturing the cell in the medium not comprisingDMSO.
 85. The method of claim 81, wherein the adalimumab preparation isformulated into a drug product if the adalimumab preparation comprises alevel of high mannose glycans at least 100% higher than a level of highmannose glycans in an adalimumab preparation produced by culturing thecell in the medium not comprising DMSO.
 86. A method of producing anadalimumab preparation having a target value of high mannose glycans,the method comprising: (a) providing a CHO cell genetically engineeredto express adalimumab; (b) culturing the CHO cell in a culture mediumcomprising 0.5% to 5% DMSO under conditions in which the CHO cellexpresses adalimumab; and (c) harvesting a preparation of the adalimumabproduced by the CHO cell that meets the target value, wherein the targetvalue is 5% to 20% high mannose glycans.
 87. The method of claim 86,wherein the target value is 5% to 10% high mannose glycans.
 88. Themethod of claim 86, wherein the target value is 10% to 20% high mannoseglycans.
 89. A method of producing an adalimumab preparation, the methodcomprising: (a) providing a CHO cell genetically engineered to expressadalimumab; (b) culturing the CHO cell in a culture medium comprising0.5% to 5% DMSO under conditions in which the CHO cell expressesadalimumab; (c) harvesting a preparation of the adalimumab produced bythe CHO cell; and (d) formulating the adalimumab preparation into a drugproduct if the adalimumab preparation comprises 5% to 20% high mannoseglycans.
 90. The method of claim 89, wherein the adalimumab preparationis formulated into a drug product if the adalimumab preparationcomprises 5% to 10% high mannose glycans.
 91. The method of claim 89,wherein the adalimumab preparation is formulated into a drug product ifthe adalimumab preparation comprises 10% to 20% high mannose glycans.92. The method of any one of claim 76, 81, 86, or 89, wherein the CHOcell is a CHO-K1 or CHO-DG44 cell.
 93. The method of any one of claim76, 81, 86, or 89, wherein the culture medium comprises 1% to 2% DMSO.94. The method of any one of claim 76, 81, 86, or 89, wherein theculture medium comprises 2% to 3% DMSO.
 95. The method of any one ofclaim 76, 81, 86, or 89, wherein the culture medium comprises 3% to 4%DMSO.
 96. The method of any one of claim 76, 81, 86, or 89, wherein theculture medium comprises 2% to 5% DMSO.
 97. The method of any one ofclaim 76, 81, 86, or 89, further comprising evaluating a level of highmannose glycans in the adalimumab preparation.
 98. A method of producinga therapeutic recombinant antibody, the method comprising: (a) providinga CHO cell population genetically engineered to express a therapeuticrecombinant antibody; (b) culturing the CHO cell population in a culturemedium comprising 0.5% to 5% DMSO, under conditions in which the CHOcell population expresses the therapeutic recombinant antibody; (c)harvesting a preparation of the therapeutic recombinant antibodyproduced by the CHO cell population; and (d) formulating the preparationof the therapeutic recombinant antibody into a drug product if thepreparation comprises a level of high mannose glycans at least 20%higher than a level of high mannose glycans present in a preparationproduced by culturing the CHO cell population in the culture medium notcomprising DMSO.
 99. The method of claim 98, comprising (d) formulatingthe preparation of the therapeutic recombinant antibody into a drugproduct if the preparation comprises a level of high mannose glycans atleast 50% higher than a level of high mannose glycans present in apreparation produced by culturing the CHO cell population in the culturemedium not comprising DMSO.
 100. The method of claim 98, comprising (d)formulating the preparation of the therapeutic recombinant antibody intoa drug product if the preparation comprises a level of high mannoseglycans at least 100% higher than a level of high mannose glycanspresent in a preparation produced by culturing the CHO cell populationin the culture medium not comprising DMSO.